Preoperative dobutamine stress echocardiography versus cardiac arteriography for risk assessment prior to renal transplantation.

Because coronary artery disease is the leading cause of death in patients with end-stage renal disease, we prospectively studied the prognostic value of dobutamine stress echocardiography (DSE) compared to coronary angiography (CA) as an evaluative tool. Thirty-three patients at high risk for coronary artery disease were selected from a cohort of 133 renal transplant candidates and underwent both DSE and CA. In this study, the value of DSE was found to exist in its strong negative predictive value (92%). A negative DSE coupled with a negative clinical cardiac evaluation was found to practicably exclude the necessity for CA. DSE can thus serve as a non-invasive, low cost screening test.

A distinct cation-sensing mechanism in MC3T3-E1 osteoblasts functionally related to the calcium receptor.

The presence of a cation-sensing mechanism in osteoblasts is suggested by the ability of specific cations to stimulate osteoblastic proliferation in culture and to induce de novo bone formation in some experimental models. Our study examines whether extracellular cations stimulate osteoblasts through the recently identified G protein-coupled calcium receptor (CaR). We found that CaR agonists, calcium (Ca2+), gadolinium (Gd3+), aluminum (Al3+), and neomycin, stimulated DNA synthesis in murine-derived MC3T3-E1 preosteoblasts, whereas magnesium (Mg2+), nickel (Ni2+), cadmium (Cd2+), and zinc (Zn2+) had no effect. With the exception of Mg2+, the cation specificities and apparent affinities were similar to that reported for CaR. CaR agonists also stimulated DNA synthesis in C3HT10(1/2) fibroblasts, but not in mesangial PVG, CHO, hepatic HTC, COS-7 cells, or malignant transformed ROS17/2.8 and UMR-106 osteoblasts. In addition, similar to other growth factors, CaR agonists activated transcription of a serum response element luciferase reporter construct (SRE-Luc) stably transfected into MC3T3-E1 osteoblasts, but had no effect on SRE-Luc transfected into CHO and COS-7 cells. We were unable to detect CaR expression by Northern analysis using a mouse CaR-specific probe or to amplify CaR mRNA by reverse transcribed polymerase chain reaction in MC3T3-E1 osteoblasts. These findings suggest that an extra-cellular cation-sensing mechanism is present in murine-derived osteoblasts that is functionally similar to but molecularly distinct from CaR.

Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts.

Extracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3-E1 osteoblasts if polyvalent cations differentially modulate the agonist-stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1 (PGE)-stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)-stimulated cAMP production. In contrast, these cations had no effect on forskolin- or cholera toxin-induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12-myristate 13-acetated (PMA) mimicked the effects of cations on PGE1- and PTH-stimulated cAMP accumulation in MC3T3-E1 cells, respectively, diminishing and augmenting the responses. Moreover, down-regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1- and enhancing PTH-stimulated cAMP production, indicating involvement of PKC-dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd(3+)-mediated suppression of PGE1-stimulated cAMP, suggesting actions independent of Gm. Thus, polyvalent cations may either stimulate or inhibit hormone-mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in vivo under different hormonal conditions.

Forskolin inhibits protein kinase C-induced mitogen activated protein kinase activity in MC3T3-E1 osteoblasts.

We recently demonstrated that stimulation of DNA synthesis in MC3T3-E1 osteoblasts involves cross-talk between protein kinase C (PKC)-dependent pathways and activation of possible nonreceptor tyrosine kinases. In the current investigation we examined whether the Raf-1/MAP kinase kinase (MKK)/mitogen-activated protein kinase (MAPK) cascade integrates cross-talk between G protein-coupled second messengers and protein tyrosine phosphorylation in osteoblasts. We investigated the effects on DNA synthesis, protein tyrosine phosphorylation, and Raf-1, MKK, and MAPK activities of PKC activation by phorbol 12-myristate 13-acetate (PMA) and of cAMP elevation by forskolin (FSK) in MC3T3-E1 osteoblasts. We found that PMA-stimulated DNA synthesis was associated with increments in tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) and activation of Raf-1, MKK, and MAPK in these cells. FSK treatment of osteoblasts, which raised intracellular cAMP levels and inhibited DNA synthesis, blocked PKC-stimulated tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) as well as inhibited PKC-stimulated MAPK and Raf-1 activities. Despite this, PMA activated the intermediate MKK step of the Raf-1/MKK/MAPK cascade in the presence of FSK. The differential inhibition of PMA-stimulated Raf-1 and MKK activities by FSK suggests that PKC activates both Raf-1-dependent and -independent pathways in MC3T3-E1 osteoblasts. Moreover, the noncoordinate effects of FSK on PMA-stimulated MKK and MAPK activities indicates the presence of a additional distal cAMP-dependent inhibitory mechanisms.

Aluminum-induced DNA synthesis in osteoblasts: mediation by a G-protein coupled cation sensing mechanism.

Aluminum (Al3+) stimulates de novo bone formation in dogs and is a potent stimulus for DNA synthesis in non-transformed osteoblasts in vitro. The recent identification of a G-protein coupled cation-sensing receptor (BoPCaR), which is activated by polyvalent agonists [e.g., gadolinium (Gd3+) > neomycin > calcium (Ca2+)], suggests that a similar physiologically important cation sensing receptor may be present in osteoblasts and pharmacologically activated by Al3+. To evaluate that possibility, we assessed whether known BoPCaR agonists stimulate DNA synthesis in MC3T3-E1 osteoblasts and examined the additive effects of Al3+ and BoPCaR agonists on DNA synthesis in MC3T3-E1 osteoblast-like cells. We found that Al3+, Gd3+, neomycin, and Ca2+ stimulated DNA synthesis in a dose-dependent fashion, achieving 50% effective extracellular concentrations (EC50) of 10 microM, 30 microM, 60 microM, and 2.5 mM, respectively. Al3+ displayed non-additive effects on DNA synthesis with the BoPCaR agonists as well as an unrelated G-protein coupled receptor agonist, PGF2 alpha, suggesting shared mechanisms of action. In contrast, the receptor tyrosine kinase agonist, IGF-I (10 eta g/ml), displayed additive proliferative effects when combined with AlCl3, indicating distinct signalling pathways. AlCl3 (25 microM) induced DAG levels 2-fold and the phosphorylation of the myristoylated alanine-rich C kinase (MARCKS) substrate 4-fold, but did not increase intracellular calcium concentrations. Down-regulation of PKC by pre-treatment with phorbol 12-myristate 13-acetate as well as PKC inhibition by H-7 and staurosporine blocked Al(3+)-induced DNA synthesis. Finally, Al3+, Gd3+, neomycin, and Ca2+ activated G-proteins in osteoblast membranes as evidenced by increased covalent binding of [32P]-GTP-azidoanilide to putative G alpha subunits. Our findings suggest that Al3+ stimulates DNA synthesis in osteoblasts through a cation sensing mechanism coupled to G-protein activation and signalling cascades involving DAG and PKC-dependent pathways.

Successful oral acyclovir desensitization.

A 65-year-old woman with the acquired immunodeficiency syndrome (AIDS) complicated by recurrent mucocutaneous herpes simplex virus (HSV) infection developed angioedema on the initiation of her second course of oral acyclovir therapy. Oral rechallenge in hospital three days later confirmed acyclovir hypersensitivity. Vidarabine and foscarnet therapies were abandoned after treatment failure and unacceptable toxicity. Acyclovir desensitization was accomplished using a protocol derived from oral penicillin desensitization regimens. Mucocutaneous HSV infection responded to intravenous acyclovir followed by chronic oral suppression without recurrences of HSV or hypersensitivity. This report is an example of acyclovir hypersensitivity and successful oral desensitization.